The Effects of Isoproterenol and Propranolol on Cytokine Profile Secretion by Cultured Tumor-infiltrating Lymphocytes Derived from Colorectal Cancer Patients

Objective: Anti-tumor immunity and cytokine profiles have important roles in the development of cancer. Norepinephrine (NE) release due to sympathetic activation leads to a Th2 deviation via the beta-2 adrenergic receptor Beta-2 adrenergic receptor (β-2AR) and could increase cancer progression. This study intends to determine the effects of isoproterenol (ISO; beta-agonist) and propranolol (PRO; beta-antagonist) on the production of IFN-γ, IL-4, and IL-17. Cytokine levels have been examined in tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMCs) of patients with colorectal cancer (CRC). The β-2AR expression on lymphocyte subsets was also assessed. Materials and Methods: In this experimental study, TILs were isolated from fresh CRC tissue and patient PBMCs were obtained just prior to surgery. The cells were cultured in medium for 72 hours. Concomitantly, cells were stimulated with 10 µg/ml phytohemagglutinin (PHA) alone or in the presence of either 1 µmol/L of PRO or 1 µmol/L ISO. The concentration of cytokines in the supernatants was measured by ELISA. Three-color flow cytometry was used to determine the expression of β-2AR on the lymphocyte subsets. Statistical analyses were performed via paired or independent t-test. Results: Levels of IFN-γ, IL-4 and IL-17 were elevated after PHA-stimulation of PBMCs and TILs. However, the elevation of IFN-γ and IL-17 production by TILs in response to PHA was significantly lower than PBMCs. In the presence of ISO, the IFN-γ/IL-4 ratio reduced in all groups, but this reduction was very low in TILs. Interestingly, the effects of PRO on cytokine production were, at least partially, comparable to those of ISO. Depressed levels of β-2AR expression were demonstrated on CD4+IFN-γ+ and CD4+IL-17+ lymphocytes in patients' PBMCs and TILs. Conclusion: This study has demonstrated the effects of ISO and PRO on cytokine production by TILs and determined β-2AR expression on these cells. ISO failed to induce a shift toward the expected Th2 cytokine profile in CRC patients' TILs, which might be due to the downregulation of β-2AR expression on TILs. Additionally, in this study, PRO induced a shift to a Th2 profile in PBMCs.


Introduction
Colorectal cancer (CRC), one of the most frequent and aggressive cancers, is the fourth leading cause of cancer deaths worldwide (1, 2). Current treatments for CRC include surgical resection (the treatment of choice), as well as radiotherapy and chemotherapy, which are curative in many patients (3). New therapeutic strategies (e.g., immunotherapy), however, are needed to improve survival (4).
Interactions between tumors and the immune system, especially in the tumor microenvironment, are dynamic, complex and bi-directional. T-helper lymphocytes (Th), which can be subdivided into 17-secreting Th17 cells, are very important in antitumor immunity. A shift toward a Th1 response results in tumor rejection, whereas a shift toward a Th2 response prevents tumor rejection (5). The role of Th17 cells in tumor immunity and their promotion or inhibition of tumor progression is unknown (6, 7). Thus, determining the role of inflammatory cells and pro-inflammatory cytokines in the tumor microenvironment of CRC is critical (8).
Two major pathway systems are involved in the neuroimmune interaction: the hypothalamicpituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS) (9). Beta2-adrenergic rebinds to norepinephrine (NE) a neurotransmitter mune cells, including natural killer (NK) cells, T tion alters proliferation, cytokine production, and the circulation of innate and adaptive immune cells es IL-10 levels in whole blood in response to lipopolysaccharides (LPS) and IL-10 production in macrophages (14,15). Thus, it seems that it has an anti-inflammatory effect. In adaptive immueven cAMP-elevating agents decrease the level of in Th2 cells (13). In addition, exposure of human IL-10. It can therefore be suggested that exposure to NE leads to a shift to a Th2-like cytokine profile or SNS activation causes immunosuppression, especially in cellular immunity (9, 16).
This study investigates the response of lymphocytes to catecholamines, and evaluates the impact of ISO and PRO on cytokine production of TILs and PBMCs derived from CRC patients.

Patients and controls
This experimental study consisted of 17 CRC patients treated at Bahman and Mehr Hospitals in Tehran, Iran, between 2009 and 2010. Patients had histologically confirmed colorectal adenocarcinoma. There were 10 male and 7 female patients, whose mean age was 56.29 years (range: 40-81 years). All patients underwent surgery . There was no history of any chemotherapy, radiotherapy, or other therapies that influenced the immune system. Patients had no autoimmune diseases.
The control group consisted of 17 healthy volunteers, of which there were 14 men and 3 women with a mean age of 36.94 years (range: 25-48 years).
The study protocol was approved by the Ethical Committee of Isfahan University of Medical Sciences. An informed consent was signed by all subjects enrolled in the present study.

Isolation of TIL
Preparation of TIL was performed by the method described by Rosenberg, with some modifications. Briefly, tissue samples were taken from the tumor mass immediately after surgical removal. Samples were placed in tubes filled with phosphate buffered saline (PBS) at 4°C and prepared for analysis within 1 hour. Fat tissues were physically removed. The specimens were then cut into small pieces and homogenized with a homogenizer. The resulting cell suspensions were filtered through sterile gauze and then a nylon mesh (100 μm) into centrifuge tubes. After several washes, the cells were placed on lympholyte-H (Cedarlane, Burlington, NC, USA) and centrifuged, without braking, for 20 minutes at 800 g and 20°C. TIL were removed from the interface and resuspended at a concentration of 1×10 5 cells/ml in complete media, which consistd of RPMI 1640 medium supplemented with 10% FBS, penicillin (100 units/ml), streptomycin (100 μg/ml), and gentamicin (40 μg/ml). Cell viability was determined by trypan blue staining.

PBMC isolation
Heparinized blood was obtained from healthy controls and patients just before surgery. Mononuclear cells were separated by the Ficoll-Hypaque gradient centrifugation technique. Peripheral blood samples were diluted with an equal volume of PBS. Cell suspensions were layered over lympholyte-H and centrifuged at 800 g for 20 minutes at 20°C without braking to separate PBMCs. Cells at the gradient interface were collected, washed twice and then resuspended in RPMI-1640. Viability and cell counts were determined.
Cells were then centrifuged for 5 minutes at 600 g, resuspended in PBS, and stained for surface antigens and intracellular cytokines. Briefly, cells were incubated with 10% human serum for 15 minutes at 4°C. The cell suspensions were labeled with fluorochrome-conjugated antibodies against cell surface antigens for 30 minutes in the refrigwas added at this stage. After washing, cells were labeled with PE donkey F(ab')2 anti-rabbit IgG as the secondary antibody for 30 minutes at 4°C in the dark. Washed cells were resuspended and 250 μl of Fix/Perm solution added for 20 minutes at 4°C. Cells were washed with 1 ml BD Perm/ Wash buffer and centrifuged. Cells were resuspended in BD Perm/Wash buffer and stained with anti-cytokine antibody for 30 minutes at 4°C in dark. Finally, cells were washed twice with 1 ml BD Perm/Wash buffer and resuspended in PBS for flow cytometric analysis. Flow cytometry was performed by FACScalibur (Becton Dickinson, Mountain View, CA), and CellQuest Pro software was utilized for data analysis.
A histogram of the fluorescence distribution of structed, and the relative geo mean fluorescence intensity (MFI) was obtained from the histogram and expressed as an index of membrane surface expression. To show lymphocyte subsets, cells were first gated by their physical properties (FSC and SSC), and a second gate was then set based on the fluorescence characteristics of the gated cells. Background staining was assessed using samples incubated with only the PE-conjugated secondary antibody.

Cell cultures, isoproterenol and propranolol treatment
PBMCs and TILs from each patient, and PB-MCs from controls were incubated in RPMI 1640 medium that contained 10% FBS and antibiotics in 24 well plates. Cells, at a concentration of 10 5 cells/well, were incubated at 37°C and 5% CO 2 for 72 hours to evaluate cytokine production. We designed four treatment conditions for the cell populations: i. cells grown in complete culture media without any stimulation, Iso or Pro; ii. cells cultured in complete culture media with 10 μg/ml PHA; iii. cells stimulated with PHA, and 1 μmol/L PRO only added at the time of activation; and iv. cells stimulated with PHA and 1 μmol/L ISO only added at the time of activation. After 72 hours, culture supernatants were collected and stored at -70°C until the cytokine assay.

Cytokine assay
by sandwich ELISA. All procedures were performed as described by the manufacturer. Briefly, standards and samples were added to 96-well cytokine coated plates. Wells were washed and detection antibody was added. Avidin-HRP and TMB substrate were used to complete the process. The wells were read at a wavelength of 450 nm by a colorimetric plate reader (Biotek Instruments Inc., Winooski, VT).

Statistical analysis
Results are presented as means ± standard deviations (SD). The paired t-test was used to measure the effect of PHA, ISO or PRO on cytokine production. The general linear model (GLM) multivariate analysis was done to see the relative effect of confounding factors on dependent variable. The analysis showed that sex and age did not have any significant effect on the variable in our model. Thus, to evaluate differences between groups, we used the independent t-test. The association was evaluated by a Pearson correlation coefficient with SPSS 16.0 software.
The levels of IL-4 in cultured PBMCs was 35.3 ± 9.2 pg/ml in the controls, 38.8 ± 16.2 pg/ml in the patient group (p=0.495), and 41.3 ± 14 pg/ml in cultured TILs, which was not significant (Table 1).
The IL-17 concentration was 1948 ± 412 pg/ml in patients' PBMCs, 615 ± 139 pg/ml in healthy controls (p<0.001), and 1795 ± 431 pg/ml? in TILs. The level of IL-17 in patients was higher than controls. and 0.1 ± 0.03 in patients' PBMCs (p<0.001). This ratio was 0.23 ± 0.07 in healthy controls' PBMCs. A positive correlation was observed in patients' PBMCs; however it was negative in TILs.

The level of IFN-, IL-4 and IL-17 cytokine production in culture supernatant in PHA-stimulated PBMCs and TILs after 72 hours
The levels of all cytokines were elevated to differing degrees in the three groups. Upon PHA stimulafold in the PBMCs of healthy controls, 20-fold in the PBMCs of patients, and 1.4-fold in TILs (Fig 1). Therefore, it seemed that the responsiveness of TILs to PHA was much lower than that of PBMCs from either patients or healthy controls.

Fig 1: IFN-production in response to PHA. HC; healthy peripheral blood mononuclear cells.
The levels of IL-4 production, in contrast, did not change upon PHA stimulation in all groups. Howfor TILs after PHA stimulation than for patients' PBMCs after PHA stimulation (14.8 ± 6.3 vs. 105 ± 37, p<0.001; Table 2).
The IL-17 concentration increased 3-fold in PHA-stimulated PBMCs in both healthy and patient samples, but only 1.5-fold in TILs.

Effects of isoproterenol (ISO) treatment on cytokine production by PBMCs and TILs
As shown in Table 1, ISO in culture medium relevel of IL-4 in all groups (Fig 2B). In addition, treatment of the cells with ISO led to a decrease in the IL-17 production in all groups ( Fig 2C). However, the in PBMCs and increased in TILs (Table 2).

Effects of propranolol (PRO) treatment on cytokine production by PBMCs and TILs
In the presence of PRO there was a significant re-duction was approximately 13-fold in both groups of PBMCs but 1.35-fold for TILs (Fig 3A).

Fig 3: A. The effects of PRO on IFN-production, B. The effects of PRO on IL-4 production, C. The effects of PRO on IL-17 production. HC; healthy control group. TILs; tumor-clear cells. PRO; propranolol.
The level of IL-4 slightly increased in the presence of PRO in patients' PBMCs and TILs (Fig. reduced in all groups, but the reduction in TILs was considerably lower than in PBMCs of patients and controls (Table 2).
In the presence of PRO, the level of IL-17 decreased by 45% in the PBMCs of healthy controls (p<0.001) and decreased 20% in TILs (p<0.001), but there was no alteration in cancer patients' PB-MCs ( Fig 3C).
in PBMCs but did not differ in TILs (Table 2).

in lymphocyte subsets
Flow cytometry was performed to evaluate the Lymphocytes were gated according to forward and side scatter, and the cell subsets were determined using CD4 (PerCp) vs. cytokine (PE) dot plots.
Th2 cells did not differ for the PBMCs of healthy sion was lower in the TILs.
were significantly higher in PBMCs of controls than in patient PBMCs and TILs.

Discussion
One of the most important functional parameters of an anti-tumor immune response is the local pro-IL-4 and IL-17, the characteristic cytokines of Th1, Th2 and Th17 cells, respectively.
higher than patients' PBMCs and 3-fold higher than controls' PBMCs. However, the level of IL-4 was not statistically different between groups. These results were consistent with a previous study in ectal tumor tissue, 84.2% in patients' PBMCs and 40% in controls' PBMCs, with no significant differences noted in IL-4 gene expression (2). A rewere higher in colorectal tumor specimens than normal tissues, but not for IL-4 mRNA (17).
It has been reported that the majority of TILs in CRC are Th1 and T cytotoxic type 1 (Tc1) cells (18). In contrast, there are reports that indicate a re-creted 20). According to research, it has been found that the Th1 immune response is protective in CRC patients (21). It seems this variation among studies could be a reflection of the heterogeneity of tumor development and/or patients' survival, even when the studies involved tumors of the same type.
Our results showed that PHA-stimulated cells produced elevated levels of all cytokines when compared to unstimulated cells, but the increase in cytokine production differed between the groups. TILs have a very low response to PHA in terms IL-4 ratio in TILs after stimulation with PHA was also lower compared to those of the other groups. This indicateda decreased shift toward a Th1 profile in TILs after PHA stimulation in comparison to the shift of patients' and controls' PBMCs. Defects in cytokine production by mitogen-stimulated lymphocytes have been reported in many conditions (22). This finding is in line with the reported decrease in PHA-induced lymphocyte proliferation in CRC (23). Because the T cell response to PHA is a complex process involving both T cells and accessory cells, we suggest that all cell and soluble costimulation, T cell receptors and signal transduction pathways should be examined in future studies.
IL-17 decreased in all cell samples, but with different reduction rates. The reduction rate was significantly lower in TILs compared to PBMCs. Our results showed that ISO increased IL-4 levels and deviation in all tested cell samples. These findings have been supported by previous observations that differentiation of CD4+T cells (12,24). It has previously been shown that naïve T cells responded to ISO, whereas effector cells were not responsive (25,26), and the pattern of TILs responsiveness to ISO in our study was somewhat similar to that of effector cells. This was consistent with several studies that have reported a higher proportion of activated cells in TILs (3,27). It was noteworthy that, in this study, Th2 deviation due to ISO was considerably slight for TILs.
We also found that ISO reduced IL-17 production in all tested groups, but at different degrees, with the largest effect on TILs. To our knowledge, our findings regarding the effects of ISO on IL-17 production in human lymphocytes was the first of its kind, but studies in mice have yielded differing results. It has been reported that epinephrine can increase serum IL-23 and IL-17 in mice (28) and murine DC exposed to epinephrine can induce IL-17A production by CD4+ T cells (11). This discrepancy could be explained by differences in the development of Th17 cells in humans and mice.
cells (15,30). Thus, it is acceptable that ISO decreases IL-17 in humans, but increases IL-17 in mice. Altogether, with regard to lymphocytes and cytokine production, it seems that ISO has a different effect in the tumor microenvironment.
els reduced and IL-4 increased in all groups (with two exceptions). Our results also showed that PRO CRC patients. In this regard, PRO seems to act as a Th2-shifting agent in cancer. As the Th1 immune response plays an important role in anti-tumor immunity, we speculate that PRO reduces anti-tumor immunity. This has recently been supported by an epidemiological study by Friedman (31). In addition, it was reported that PRO reduces cytokine secretion by the hypertrophic scar cells (32). In contrast, Fitzgerald has suggested that PRO, as an NE receptor blocker, may suppress cancer cell development (33). This discrepancy might be due to different aspects of the studies. Some focus on the immunological view and cells, whereas others pursue cancer cell development. Of course, it is clearly known that the anti-tumor effects of PRO are not due to beta-AR antagonism. PRO can inagonist for adrenoceptor (34, 35), which might be responsible for the abovementioned effects of PRO on cytokine production.
An important finding in our study is the downreg-phocytes in patients' PBMCs and TILs. In keeping sion or sensitivity on PBMCs has been observed in rheumatoid arthritis (RA) patients, patients with septic shock, and those exposed to stressful conditions such as caregivers (36,37). It has been responses toward a Th2 paradigm (10, 16). Theredemonstrates a failure to induce a shift toward a Th2 cytokine profile. The ability to generate a Th1 type response is critical for the optimal function of anticancer lymphocytes (5). Taken together, it is a way to promote anti-tumor immune response. Interestingly, our results have shown that the shifting of TILs toward Th2 due to ISO is lower than that of patients and controls' PBMCs. The downconsidered as one of the mechanisms.
One of the limitations in the current study are the relatively small case numbers in each group. Therefore, we believe that the future studies with larger case number are needed to confirm the findings of the current study.

Conclusion
This study demonstrated the effects of ISO and PRO on cytokine production by TILs and These findings showed a failure of ISO to induce a shift toward a Th2 cytokine profile in CRC patients' TILs. This failure might have been TILs. We also demonstrated a Th2 shift in CRC patients' immune cells upon PRO treatment. The results further indicated that ISO reduced IL-17 production by human PBMCs.